This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Multidrug efflux pumps interfere significantly with cancer chemotherapy and the treatment of bacterial infections, by recognizing a number of structurally unrelated toxic compounds and actively extruding them from cells. Our long-term goal is to elucidate the structures and fundamental mechanisms that give rise to multiple drug recognition and extrusion in these multidrug transporters. The primary target of this proposal is the Escherichia coli AcrB transmembrane efflux pump, which shows the widest substrate specificity among all known multidrug transporters, ranging from most of the currently used antibiotics, disinfectants, dyes, detergents, to simple solvents. We have determined the x-ray structures of AcrB in the presence of four structurally different agents. The crystal structures illustrate that three ligand molecules bind simultaneously to the extremely large central cavity of 5000 cubic Angstroms, primarily by hydrophobic, aromatic stacking and van der Waals interactions. Each ligand uses a slightly different subset of AcrB residues for binding. The bound ligand molecules often interact with each other, stabilizing the binding. The subsequent study of the efflux pump by crystallizing a mutant AcrB with five structurally diverse ligands indicates that AcrB consists of two distinct binding sites. These five ligands not only bind to various positions of the central cavity, but also to residues lining the deep external depression formed by the C-terminal periplasmic domain. The structures also suggest that AcrB assembles as a trimer of three identical channels for the extrusion of drugs. Each subunit of AcrB in the trimer forms its own channel for multidrug transport. The specific aims are to: 1. identify important residues for multidrug binding in AcrB, 2. examine the mechanism of multidrug transport in the efflux pump, 3. determine the x-ray structure of the AcrAB co-crystal complex.